1b.Approach (from AD-416)
Maize is an important crop as well as a model organism for other cereals such as sorghum, barley, rice and wheat. Our long term goal is to identify and characterize the activity of maize genes involved in plant production including tolerance to stressful growth conditions and regulation of flowering time. Recent work in model systems demonstrates that the circadian regulation of physiological activities is required for optimal plant growth and for tuning of responses to environmental cues. A comprehensive understanding of the circadian system in cereals is lacking; therefore, this proposal seeks to define the maize circadian system and assess the circadian oscillator’s contribution to important agronomic traits. Known circadian mutants will be tested for their response to salt and osmotic stress. Genes under circadian regulation in cereals will be identified by expression profiling, and this information used to computationally predict regulatory DNA elements that contribute to circadian gene expression. Reverse genetic approaches will evaluate the role of candidate photoperiodism genes in determining the timing of maize flowering. Maize inbreds and recombinant inbred lines will be analyzed for natural variation in overt circadian rhythms. DNA sequences, genes, mutants, and inbred lines identified here provide two types of tools: a better understanding of fundamental processes in environmental responses and targets that can be used to improve crop productivity.

3.Progress Report
The endogenous circadian clock manages plant growth, flowering time, and stress responses by organizing plant physiology. A comprehensive knowledge of the circadian system in cereals holds great potential to provide novel avenues for optimization of important agronomic traits and improve crop production. The research group’s goal is to define the genetic makeup of the maize circadian system and assess its impact on key cereal agronomic traits.

Work completed this year is leading toward an understanding of the genetic framework behind the maize circadian system. Progress comprises identification of a substantial number of maize transcripts under circadian regulation and discovery of several likely maize clock components. Microarray analysis showed 10% of maize transcripts exhibit a circadian rhythm; a clear illustration of the maize circadian oscillator’s broad transcriptional regulatory purview. Important agronomic findings were that circadian regulation coordinates expression of genes contributing to key metabolic processes like photosynthesis, starch utilization, and biosynthesis of phytohormones, cell walls, and several secondary metabolites. These findings will inform maize researchers of the specific areas in maize physiology where circadian regulation is important. Also, this work highlights circadian regulatory circuits that can be harnessed to direct coordinated regulation of the principal gene sets underlying several important physiological processes. Five potential maize clock genes were tested for circadian clock function by expressing the transcript of each separately to high levels in Arabidopsis. Transgenic Arabidopsis plants expressing the maize proteins exhibited circadian clock-related phenotypes matching those caused by high expression levels of the Arabidopsis orthologs; thus, these maize proteins are likely to be important molecular components of the maize circadian clock. Also this work confirms these genes are good targets for genetic dissection of the maize circadian system. The ongoing collaboration with Pioneer Hi-Bred delivered four potential mutant lines in the maize GIGANTEA (ZmGI) gene. The position of these mutations was confirmed by sequencing and genetic crosses were initiated to commence genetic studies of these alleles.

A long-term project initiated in 2009 is designed to identify the most potent flowering time genetic loci from the day length insensitive B73 inbred and the photoperiodic CML333 inbred. The basic approach is to develop a series of recombinant inbred lines (RIL) where B73 genetic material incorporated into CML333 produces a day length insensitive plant. Similarly, CML333 loci causing B73 to a gain of photoperiodic behavior with be genetically mapped in RIL. To start, an F2 population was screened for flowering time and several individuals were found to display the exact flowering phenotype of the parental type. These individuals were advanced to the next generation to begin the genetic analysis needed to pinpoint the loci associated with the modified flowering time behavior.

4.Accomplishments
1.
Circadian profiling of maize transcription. The extent to which the circadian clock regulates gene expression in maize was not known. ARS scientists in Albany, CA determined genome-scale circadian regulation of gene expression was determined by ARS scientists in Albany, CA in the temperate maize inbred B73. Gene expression was evaluated for ~13,000 unique genes. Rhythmic expression was exhibited by 12% of these genes, including those involved in energy metabolism, nutrient assimilation, cell wall biosynthesis, and synthesis of plant hormones and other secondary metabolites. This work provided a framework for understanding the far-reaching influence that the circadian clock has over many aspects of maize physiology that ultimately impact crop production.

2.
Preliminary characterization of novel mutations in a gene needed to produce correct circadian rhythms in maize. The genetic factors involved in circadium rhythium in corn are not well understood. Four different mutations in a gene likely to be critical for maize plants to produce circadian rhythms of the correct length were confirmed by ARS scientists in Albany, CA. An industrial cooperator, furnished the lab with the seeds for these mutants through a CRADA previously established for project 5335-21000-025-00D. The lab found the site of each mutation within the maize genome with DNA sequencing and initiated genetic crosses to make plant lines for future characterization of each mutant. Future testing will be focused on evaluating whether the mutations in this gene change maize traits associated with the circadian clock, including the timing of flower development. This work will provide insight into the relationship between circadian rhythms and important agronomic traits in maize and cereals in general.

3.
Functional characterization of maize circadian clock genes. The molecular function of potential maize circadian clock genes was not known. ARS scientists in Albany, CA tested them by overexpression in the model plant Arabidopsis thaliana. The DNA sequences encoding several of the likely maize circadian oscillator components were cloned and confirmed, including those for ZmGI (flowering time/clock), ZmZTL (clock), ZmTOC1 (clock), ZmCCA1 (clock), and ZmCKB3 (clock). Transgenic expression of each maize sequence at high levels in Arabidopsis produced effects on circadian rhythms and flowering time that were highly comparable to the consequences of overexpression of the native Arabidopsis genes. This line of experimentation demonstrated these maize clock genes posses the functional attributes of their Arabidopsis counterparts. Now that circadian oscillator genes have been found in maize, these will serve as tools that can be used to test the extent to which circadian rhythms influence maize agronomic traits.